Purification and characterizations of phytase enzyme form aspergillus niger

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MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY PURIFICATION AND CHARACTERIZATIONS OF PHYTASE ENZYME FORM Aspergillus niger SIZE 14-15 SUPERVISOR STUDENT Dr. DUONG THI HUONG GIANG NGUYEN CONG DANH Student code: 4084238 Session: 34 Can Tho, 2013 APPROVAL SUPERVISOR Dr. DUONG THI HUONG GIANG STUDENT NGUYEN CONG DANH Can Tho, May 10, 2013 PRESIDENT OF EXAMINATION COMMITTEE Abstract The aim of this study was to purify and characterize an enzyme phytase obtained from Aspergillus niger isolate. The results showed that extracellular phytase from Aspergillus niger can be purified by ammonium sulfate precipitation at 80-90% saturation in combination with cation-exchange chromatography on SP-streamline following hydrophobic interaction chromatography on Phenyl-Sepharose. Phytase purification fold was 12.56 and activity recovery was 48%. SDS-PAGE revealed that the purified phytase behaved as a protein with molecular mass of about 87kDa. Optimum phytase activity was at 65°C in the presence of 40mM Ca2+. Key words: Aspergillus niger, hydrophobic interaction, Cation-exchange chromatography, SP-streamline, Phenyl-sepharose, phytase, phytate. i CONTENTS Abstract i Contents ii I. Introduction 1 II. Materials and methods 2 2.1 Materials 2 2.2 Methods 2 III. Results and Discussion 8 3.1. Extraction of phytase from A. niger fresh biomass 8 3.2. Purification of phytase 8 3.2.1. Precipitation with ammpnium sulphate saturation 8 3.2.2. Purification of phytase by Cation exchange 9 chromatography 3.2.3. Hydrophobic interaction chromatography 11 3.3. Characterization of phytase 3.3.1. The effect of ion Ca 2+ 15 on phytase activity 15 3.3.2. Temperature optimum of phytase activity 16 IV. Conclusions and Suggestions 18 4.1. Conclusions 18 4.2. Suggestions 18 References 19 ii I. Introduction Phytate (myo-inositol hexakisphosphate) is the primary storage form of phosphorus in plants. They occupy about 1-3% of the seeds of grains and legumes, and 60-80% of the total amount of plant phosphorus (Nelson, 1967). Phosphorus is essential for feeding animals, poultry and even for human diet. It is supplied in the form of phytate or phytic acid. Naturally, phytases are known as a group of enzyme able to catalyse the hydrolysis of phytate to free phosphate, a form of phosphorus that is easily to be absorbed in the animal digestive tract. Unfortunately, monogastric animals and human are lacking in phytase. As a result, phosphorus from phytate can not be absorbed and therefore it is excreted in the feces that cause the environment pollution (Mullaney et al., 2000). For this reason, supplementing of phytase in the feed or human diet will help to solve the problem. Phytase can be obtained from many sources such as bacteria (Bacillus, Enterobacteria), filamentous fungi (Aspergillus sp., Penicillin sp., Mucor sp.). Among them, A. niger is a preferable species for exploration due to its high phytase production. Recently, an A. niger strain which was able to give high phytase production was isolated in the Lab of Enzyme Technology, Biotechnology R&D Institute (Nguyen Nhat Khoa Tran, 2012). As the continuation of this research, the thesis on "Purification and characterizations of phytase enzyme from Aspergillus niger" has been performed with the aim to obtain purifed phytase and some its valuable characteristics to aplly in feed or food industries. 1 II. Materials and Methods The thesis was done from 12/2012 to 04/2013 in the Laboratory of Enzyme Technology, Biotechnology R & D Institute, Can Tho University. 2.1. Materials The A. niger isolate was supplied by BSc Trần Nguyễn Nhật Khoa (2012). Equipments: - Mini protein II (Bio Rad ). - Chromatography system (Bio Rad). - Centrifuge Eppendorf (Germany). - Spectrophotometer (Japan). - Refrigerated Centrifuge (Germany) - Other lab facilities. Chemicals: Bovin Serum Albumin (BSA) (Merck), Tris-HCl (Sigma), Trichloroacetic acid (TCA) (Merck), Casein (Prolabo), Acrylamide (Bio Rad), Sodium hydroxide (NaOH) (Merck), Sodium Dodecyl Sulfate (SDS) (Sigma), Bromophenol blue (Merck). Glycine (Merck). SP-Streamline gel (GE Healthcare). Phenyl Sepharose (GE Healthcare), Folin solution, Bradford solution, Glucose, Sucrose, Malt extract, KH2PO4, KCl, MgSO4.7H2O, NaCl, CaCl2.2H2O, MnSO4.4H2O, FeSO4.7H2O (China). 2.2. Methods 2 2.2.1. Extraction of phytase from A. niger fresh biomass 2.2.1.1. Collection of crude phytase extract Aim: Extracting crude phytase from A. niger fresh biomass for purification. Procedure: A. niger was cultured on the semi-solid medium containing 30g husk + 60g corn powder + 50ml supplement solution including Glucose (5g/L), Sucrose (5g/L), Malt extract (5g/L), KH2PO4 (1g/L), KCl (0.5g/L), MgSO4.7H2O (0.1g/L), NaCl (0.1g/L), CaCl2.2H2O (5g/L), MnSO4.4H2O(0.01g/L), FeSO4.7H2O(0.01g/L). Crude phytase solution was extracted accordingly to the following scheme. Semi-solid culture medium Inoculate 2ml spores (108 CFU/ml) five days incubation at 30°C Extraction of phytase from fresh mold biomass by glycine-HCl 0.2M, pH 3.5 Centrifugation at 7000 rpm, 20 mins, 4oC Remove particles Crude phytase extract Protein concentration was measured by Bradford method (1976) and phytase activity was determined by the method of Heinonen and Lahti (1980). 3 2.2.1.2 Studying the effect of Ca2+ ion on phytase activity Experimental design: Completely random with a factor: the concentration of Ca2+ varied from 0, 5, 10, 20, 30, 40 to 50mM. The experiment was repeated three times with 7 treatments. Totally, there were 21 experimental units. Experimental performance: CaCl2 salt was added in crude phytase extract to achieved different concentrations as mentioned above. Phytase activity was determined by Heinonen and Lahti method (1980). Evaluation criteria: phytase activity (U / mg protein). 2.2.1.3. Purification of phytase by ammonium suphate precipitation Crude phytase was precipitated from the extract with amonium sulfate 80% saturation for 1-2 hour(s) at 4°C. Then, the precipitated protein was dialyzed against buffer glycine-HCl 0.2M, pH 3.5 Protein concentration was measured by Bradford method (1976) and phytase activity was determined by the method of Heinonen and Lahti (1980). 2.2.1.3. Purification of phytase by cation exchange chromatography The enzyme solution after dialysis was aplied onto the cation-exchange column SP-streamline. 4 The chromatography column was equilibrated with the buffer glycine-HCl 0.2M pH 3.5. The protein solution was loaded into the column at the rate of 0.8ml/minute. The column was washed with the same buffer to remove the unbound protein. Then, bound phytase was eluted with gradient NaCl from 0 to 0.5M. Obtained protein fractions were determined protein concentration by Bradford (1976) and phytase activity by Heinonen and Lahti method (1980). Fractions with enzyme activity were checked for purity on SDS-PAGE. Then they were further dialyzed against glycine-HCl 0.2M containing 30% ammonium sulphate, pH 3.5 and loaded onto the hydrophobic interaction column. 2.2.1.4 Purification of phytase by hydrophobic interaction chromatography Hydrophobic interaction column was equiblirated with glycine-HCl 0.2M pH 3.5 containing 30% AS saturation. The enzyme fractions obtained from the ion-exchange column was loaded into this column at the rate of 0.8ml/minute. Bound proteins were eluted with gradient ammonium sulfate saturation from 30% to 0%. Protein fractions were collected and measured by Bradford method (1976). Phytase activity was determined by Heinonen and Lahti method (1980). The purity of the enzyme was checked on SDS-PAGE. 5 The purification process of phytase from A. niger fresh biomass was performed by the following procedure: A. niger fresh biomass homogeniztion with glycin-HCl 0.2M, pH 3.5 Crude phytase extract Saturated ammonium sulfate 80% precipitation Ion exchange chromatography Hydrophobic interaction chromatography SDS-PAGE electrophoresis SDS-PAGE electrophoresis Pure phytase 2.2.2. Studying the optimal temperature of phytase activity The experimental design: Completely random with a factor: temperature of the reaction 35°C, 45°C, 55°C, 65°C, 75°C, 85°C, 95°C . The experiment was repeated three times with 7 treatments. In totall there was 21 experimental units. Experimental performance: The phytase hydrolysis reactions on synthetic phytate as a substrate were performed at different temperature as mentioned above. Phytase activity was determined by Heinonen and Lahti method (1980). Evaluation criteria: phytase activity (U / mg protein). 2.3. Statistical analysis method 6 Microsoft Excel software version 2003 and Statgraphic software version 15.0 were used to analyze the experimental data. 7 III. Results and Discussion 3.1. Extraction of crude phytase from A. niger fresh biomass Fresh A. niger biomass 900g was homogenized with 1000ml Glycine-HCl 0.2M pH 3.5 buffer. The mixture was centrifuged to obtain the crude phytase solution. The volume of crude phytase extract were obtained about 700ml. There was 498.71mg protein obtained with specific activity about 0.141U/mg. 3.2. Purification of phytase 3.2.1. Precipitation with ammpnium sulphate saturation Crude phytase solution were precipitated with 80% ammonium sulfate saturation (Đỗ Thị Thu Trang, 2011) in 2 hours at 4oC. The protein precipitate was redisolved in GlycineHCl 0.2M pH 3.5 and dialyzed against the same buffer to remove ammonium sulfate. The specific phytase activity after dialysis was about 0.770U/mg. It was 5.44 fold higher comparing with the crude enzyme extract. SDS-PAGE was performed to check for the purity of this protein fraction. SDS-PAGE electrophoresis showed that after precipitation some contaminated proteins were removed, however the enzyme impurity was still high, so it was needed to purify it further by other methods. 8 116kDa 66,2kDa 45kDa 35kDa 25kDa 18,4kDa 1 2 3 Figue 5 . SDS-PAGE of ammonium sulfate preparation of phytase. 1. Standard protein; 2. Crude phytase extract; 3. Ammonium sulfate protein preparation 3.2.2. Purification of phytase by Cation exchange chromatography Phytase from A. niger was rather stable at low pH and its pI was about 4.7. Thus, the cation exchange chromatography at pH 3.5 were used to purify phytase. 9 The enzyme fraction obtained from ammonium sulphate precipitation was loaded onto cation exchange column SPStreamline. 4.5 0.6 4 OD 280nm 3 0.4 2.5 0.3 2 1.5 0.2 1 NaCl concentration (M) 0.5 3.5 0.1 0.5 0 0 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 Protein fractions Figue 6. Chromatogram of ammonium sulphate phytase preparation on SP-Streamline cation exchange column. The chromatogram of ammonium sulphate preparation on SPStreamline column (Figure 6) showed that there was only one protein peak eluted at 0.116M-0.256M NaCL. This protein had specific phytase activity about 1,154(U/mg), which was 1,5 fold and 8.16 fold purity, higher than the specific activity of ammonium sulphate enzyme preparation and crude enzyme extract respectively. SDS-PAGE (Figure 7) showed that after cation exchange chromatography SP-Streamline, several contaminated proteins in the phytase fraction have been removed (lane 4). It has been partially purified in camparison with the ammonium sulphate preparation (lane 3). However, there were still four protein bands in this fraction so it should be further purify to get the 10 homogenous form. For this reason, hydrophobic interaction chromatography on Phenyl Sepharose column was aplied. 116kDa 66,2kDa 45kDa 35kDa 25kDa 18,4kDa 1 2 3 4 Figue 7. SDS-PAGE of enzyme fraction after SP-Streamline cation exchange chromatography. 1. Standard protein; 2. Crude phytase extract; 3. Saturated ammonium sulfate 80% precipitation sample; 4. Enzyme fraction eluted at 0.116M0.256M NaCL. 3.2.3. Hydrophobic interaction chromatography The hydrophobic interaction chromatogram of phytase fraction after SP-streamline column has been shown on the Figure 8. There were two main protein peaks. Peak FI had no phytase activity. Peak FII was the enzyme phytase with specific acticity of 1,778U/mg, it was 12,56 fold purified incomparison with crude phytase extract. 11 AS concentration (%) 4 35 3.5 30 3 25 OD 280nm 2.5 20 2 15 1.5 10 1 5 0.5 0 0 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 106 111 116 121 126 131 136 Protein fractions Figure 8. Hydrophobic interaction chromatogram on Phenyl Sepharose of Phytase fraction after SP-Streamline column SDS-PAGE (Figure 9) revealed the homogenous phytase with one major protein band of 87kDa (lane 5). This result was similar to results of Greiner et al. (2009), the molecular mass of extracellular phytase from A. niger 11T53A9 was about 85kDa. This result is in accordance with the research by Ashok Pandey et al. (2001) which showed phytase of some Aspergillus strains had molecular mass from 40 to 100kDa. Sariyska et al. (2005) found an extracellular phytase from wild strain A. niger with a low molecular mass 39kDa. In addition, the molecular mass of the phytases from bacteria is also low. For example, extracellular phytase from Bacillus sp. DSll was 44 kDa (Young-Ok Kim et al., 1998) and alkaline phytase from Lilium longiflorum pollen grain was 52-55 kDa (Barry G. et al., 2006). 12 87kDa 116kDa 66,2kDa 45kDa 35kDa 25kDa 18,4kDa 1 2 3 4 5 Figue 9. SDS-PAGE of phytase fractions after hydrophobic interaction chromatography on Phenyl Sepharose. 1. Standard protein; 2. Crude phytase extract; 3. Ammonium sulfate 80% precipitate; 4. Phytase after SP- Streamline column; 5. Phytase after Phenyl Sepharose column. Table 2 showed the purification scheme of the enzyme phytase from A. niger isolate. The phytase was purified by three steps: ammonium sulfate precipitation at 80% saturation, cation exchange chromatography on SP-Streamline and hydrophobic interaction chromatography on Phenyl sepharose. The enzyme yield was 3.85% with 48% recovery activity. Specific activity was 12.56 fold higher than crude phytase extract from the fresh A. niger biomass. 13 Table 2. Purification scheme Purification steps Crude extract Protein Total (mg) Yield (%) Activity Specific Purifi- Total Recovery activity cation (U) (%) (U/mg) (fold) 498.31 100 58.117 100 0.141 1 47.339 11.53 36.437 63 0.770 5.44 27.598 6.72 31.846 55 1.154 8.16 15.813 3.85 28.112 48 1.778 12.56 Ammonium sulfate 80% precipitation SP-Streamline Phenyl Sepharose In comparison with the other purifcation procedures such as Sariyska et al. (2005), who purified phytase from A. niger wild species by three steps: PS 50 membrane filtration, Sephadex G-100 gel filtration chromatography column and DEAESepharose CL 6B ion exchange chromatography column, and Greiner et al. (2009) purified phytase from A. niger 11T53A9 through these steps of ammonium sulfate precipitation from 090% saturation, four ion exchange chromatography column and gel filtration chromatography (DEAE Sepharose CL 6B, CM Sepharose CL 6B, Sephacryl S -200 HR and Mono S HR 5/5), the purification procedure in this thesis was rather simpler and the enzyme preparation was purer. In general the purification procedure for phytase from A. niger can be established as in the following scheme: 14 Crude phytase extract from fresh A. niger biomass Saturated ammonium sulfate 80% precipitation Dialysis against glycine-HCl 0,2M pH3,5 buffer solution Ion exchange chromatography on SP-Streamline Eluted with NaCl 0-0,5M Eluted enzyme fraction SDS-PAGE Hydrophobic interaction on Phenyl Sepharose gel Eluted with ammonium sulfate 30%-0% Eluted Phytase faction SDS-PAGE Pure phytase Figure 10. Purification procedure of Phytase from A. niger isolate 3.3. Characterization of purified phytase 3.3.1. The effect of ion Ca2+ on crude phytase activity. Figue 11 demonstrated that crude phytase activity was affected by Ca2+ ion. The presence of ion Ca2+ from 5- 50mM enhanced the enzyme activity and the specific activity was significantly different compared to control (Table 4, Appendix 2). Adding 40mM Ca2+ increased specific activity of phytase 74,07% comparable to the control (without Ca 2+). However, 2+ when the concentration of Ca increased more than 40mM, the enzyme activity decreased, possibly high concentration of phytase inhibit the enzyme action. 15 Specific activity (U/µg) 160 b 140 120 100 a c d d 5 10 b e 80 60 40 20 0 0 20 30 40 50 Concentration of Ca2+ (mM) Figue 11. The effect of Ca2+ion on phytase activity Also, Sariyska et al. (2005) reported that crude phytase from A. niger wild type was active in the presence of 0.1mM Ca2+. Phytase from Bacillus sp. DS11 active in the presence of 5mM Ca2+ (Young-Ok Kim et al., 1998). 3.3.2. Temperature optimum of phytase activity Temperature is an important factor directly affecting the catalytic activity of the enzyme. Almost all enzymes were denatured under high temperature due to the disruption of enzyme structure, except some thermophylic enzymes. The results in Figue 12 showed that the phytase from A. niger isolate was active in a range of 45 oC to 95 oC, and the optimum temperature was 65oC. It seemed this phytase is thermotolerant. Similarly, Kim et al., 1998 reported that the active temperature of different phytase was in a range of 40 oC - 77 oC. Also, Sariyska et al. (2005) showed that the extracellular phytase from wild Aspergillus niger exposed the optimum temperature at 55°C. 16
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